Tyrosine and Glutamine Mutants

In a second set of targeted mutations, it was aimed to replace the aforementioned isoleucine with a tyrosine or glutamine. The mutations were only carried out to replace the isoleucine in the first subunit, since alteration of both positions seemed to have a similar effect. The tyrosine was expected to mimic the phenylalanine in size, while also adding a polar group, which should alter the interaction between the polar nicotinamide molecule and the amino acid residue. A glutamine was introduced, since it is structurally similar to the initially present isoleucine, carrying a polar group but no charge, which also should alleviate the mutation’s effect on the sensor’s affinity. The cDNA of the isoleucine mutants (I189Y and I189Q) was constructed and the protein was heterologously expressed. The purified protein was investigated by fluorescence spectroscopy. The data for the NADH titrations is shown in Figure 35.

Figure 35 Fluorescence emission maxima at 515 nm after excitation at 490 nm and divided by the fluorescence maximum obtained after excitation at 590 nm are plotted against the applied NADH concentrations for the Peredox-mCherry mutants. Panel A shows the titration experiment results for the I189Q mutation (mutation in the first T-Rex subunit, SU1). Panel B shows the titration experiment

results for the I189Y mutation (mutation in the first T-Rex subunit, SU1). The spectra were normalized to the maximum value in absence of NADH. The plot of relative fluorescence against NADH concentration was fitted by a Hill equation (dashed curves) and the parameters are given in the inset. Data are mean values ± S.D. of 3 experiments.

The determined affinities for both mutants were decreased compared to the parental Peredox protein (~5 nM) (Hung et al., 2011), since the KD of the I189Q mutant for NADH was determined to be about 21 nM (Figure 35 A), while the affinity for the tyrosine variant was even further lowered to 79 nM (Figure 35 B).

The Hill factor is close to two for both mutants and therefore in accordance with the determined Hill factor for Peredox-mCherry, showing high cooperativity of NADH binding. The dynamic range of the sensor mutants, however, is slightly reduced from 2.5 for the original Peredox sensor to about 1.5 in both mutants.

These mutants, though demonstrating reduced affinities towards NADH, are still not optimally suited for the application in bacterial cells yet. Due to the cytosolic NADH concentrations in the micromolar range (Bennett et al., 2009; Tejwani et al., 2017), a potential sensor should demonstrate an affinity towards the desired analyte in the same order of magnitude.

In summary it was shown that the exchange of either the nonpolar and aliphatic amino acid isoleucine at position 189 in the first subunit of T-Rex, or the exchange of the nonpolar and aromatic amino acid phenylalanine at position 194 in the second subunit of T-Rex by the negatively charged glutamic acid leads to a sharp decrease in the sensor’s affinity towards NADH. This indicates that both of these positions are crucial for tuning the sensors affinity towards NADH. The negatively charged amino acid residues in the mutants might lead to too strongly repulsive forces between the protein and the negatively charged nucleotide, and, therefore, reduce the affinity of the mutants towards values in the millimolar range.

Therefore, in a second set of experiments, it was aimed to exchange the nonpolar aliphatic amino acid by tyrosine, a close homologue to phenylalanine, albeit a polar aromatic amino acid, and also by glutamine, a polar but uncharged amino acid.

Titration studies with these mutants showed affinities were lowered compared to the parental Peredox-mCherry. However, for an application in bacteria, the

106 4 Adjusting Peredox-mCherry for Usage in High NADH Concentration Environments

affinities were not lowered sufficiently, indicating that the structural or electrostatic changes in the NADH binding environment are too subtle to change the binding behavior of the sensor into the micromolar range as is desired for application in high NADH concentration environments. Hence, the generated Peredox variants do still not appear appropriate for intracellular NADH sensing in R. eutropha.

5 New Sensors Based on NIR Probes

The design of a new NADH-sensor for application in bacterial cells, must meet certain conditions, in order to generate an optimized sensor reporting on the chosen analyte. In case of a quantitative NADH sensor designed for in vivo monitoring in bacterial cells based on the experience with previously available NADH sensors, the following points should be fulfilled:

1. Affinity towards NADH in the high micromolar range (KD ~ 100 M)

2. Negligible affinity towards structurally related molecules such as NAD⁺, NADPH, etc.

3. NADH-dependent fluorescence response in the red region of the spectrum 4. Large dynamic range

5. Ability to normalize the fluorescence signal, for better intercellular comparability

6. No pH effects on sensor response

7. Robust expression of the sensor in various prokaryotic, but possibly also, eukaryotic cell hosts

The utilization of a red-fluorescent protein appears as an optimal choice for the fluorescent probe, in order to benefit from the aforementioned advantages. For a NADH-dependent fluorescence response in the red spectral range, a suitable fluorophore with the right spectral characteristics should be incorporated into the sensor. iRFP713 is a far-red emitting protein derived from a bacterial phytochrome of Rhodopseudomonas palustris (RpBphP2), which hence binds biliverdin IX as a cofactor (Filonov et al., 2011). In a 2013 paper, the group around Verkhusha described iSplit, a probe for protein-protein interaction (PPI) derived from this far-red fluorescent protein iRFP713. In this probe, the subdomains of the chromophore-binding domain (PAS and GAF domain) are split and separately expressed, with the remarkable observation that a red fluorescent protein is formed upon recombination of the domains mediated by protein-protein interactions (Filonov & Verkhusha, 2013).

This iSplit probe appears as a good starting point to develop concepts for the construction of new infrared sensor proteins.